Summary of work: Mitochondrial dysfunction may play an important role in the aging process or in age-associated neurodegenerative diseases. Dysfunction may be related to mtDNA damage that accumulates with age or to other properties that are independent of mtDNA-encoded genes but are critical to maintenance of function, especially during stress or conditions that lead to necrotic or apoptotic death. We have continued to develop competitive PCR reagents for use in measuring "common deletions" that occur across species, including mice and primates. These studies are onging and incomplete. Studies were continued on cell death in PC-12 cells induced by mitochondrial poisons that may cause either necrotic or apoptotic cell death. NGF and a cyclic AMP analogue significantly attenuate cell death during exposure to these agents. The necrotic versus apoptotic nature of the cell death produced by these inhibitors remains under investigation along with with the effect of overexpression of the antiapoptotic, mitochondrially associated protein Bcl-XL. Bcl-XL substantially attenuates cell death induced by rotenone. Clones expressing different levels of this protein are being evaluated for effectiveness and relationship to redox state, particularly GSH. Additional studies were begun using fluorescent probes to study generation of reactive oxygen species (ROS). The mitochondrial inhibitor antimycin A increased superoxide generation as much as 4-fold, an effect that could be blocked by myxothiazol, clearly showing mitochondria to be the source; no effect was found from Bcl-XL overexpression. Additional studies were begun on human neuroblastoma SH-SY5Y parental and Alzheimer's Disease cybrid cells (they contain mitochondria derived from AD platelets) obtained from MitoKor. A cybrid mostly homoplasmic for an AD-associated mutation in cytochrome oxidase I appears more sensitive to the calcium-elevating, cytotoxic agent thapsigargin. Understanding the mechanisms by which partial mitochondrial dysfunction can lead to cell death, particularly apoptotic cell death, may be important in understanding the role of mitochondrial dysfunction in neurodegeneration and age-associated changes.